Method for regulating retinal endothelial cell viability

ABSTRACT

Here provided is a method for regulating retinal endothelial cell viability in a mammal by administering to the mammal a therapeutically effective amount of a quinic acid analog. The method may be applied to prevent, treat or cure pathological conditions of retinal endothelial cells associated with radiation retinopathy, diabetic retinopathy and chemotherapy for retinoblastoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of earlier-filed U.S.Provisional Patent Application Nos. 61/723,733 filed on Nov. 7, 2012 and61/807,303 filed Apr. 1, 2013, and is a continuation-in-part of U.S.patent application Ser. No. 13/320,540 filed on Jun. 6, 2012, thedisclosure of which applications are incorporated herein by reference.

GOVERNMENTAL SUPPORT

Certain aspects of the invention, namely, the study on radiationretinopathy in Example 1, were supported by NIH/NIAID grantR33AI080534-01. The U.S. government may have certain rights in theinvention.

FIELD OF INVENTION

The present invention relates to a new use of quinic acid analogs inregulating retinal endothelial cell (REC) viability. More specifically,the invention relates to methods to prevent, treat or cure diabetes-,radiation-, or chemotherapy-induced pathological conditions in RECs.

BACKGROUND

Retinal neovascularization (RNV) represents the leading cause ofblindness in humans and can be triggered by a variety of ocular insults,e.g., high glucose, radiation, and chemotherapy. RNV may be caused byREC death due to an exuberant pro-inflammatory response triggered bydamages to the posterior vascularized portion of the eye (Brown et al.,1982; Viebahn et al., 1991; Zamber and Kinyoun, 1992). Because newvessels formed under hypoxia are fragile and leaky, RNV often leads tomacular edema, retinal detachment and blindness. For example, as shownin FIG. 1, irradiation of the eye can trigger leukocyte adhesion,accumulation, and blockage of retinal vasculature, which may result inhypoxia, abnormal retinal neovascularization and ultimately loss ofvision.

Treatments for RNV include glucocorticoids, anti-vascular endothelialgrowth factor (VEGF) monoclonal antibodies, and surgical intervention(panretinal photocoagulation [PRP]) (Aiello et al., 1995; Googe et al.,2011). These treatments are often not effective and also limited by sideeffects such as ocular hypertension, glaucoma, cataracts, retinaldetachment, and endophthalmitis (Conti and Kertes, 2006; Gillies et al.,2006; Waisbourd et al., 2011).

We have discovered a new class of quinic acid analogs (QAAs) that areare resistant to bacterial degradation (Zeng et al., 2011; Zeng et al.,2009). We have previously demonstrated that the QAAs, e.g., KZ-41(1,3,4,5-tetrahydroxy-1-cyclohexanecarboxylic acid), can exert asignificant pro-survival effect in a whole murine model of high doseradiation injury. See U.S. Patent Application Publication No.2012/0283331. Here we disclose another surprising discovery that QAAscan regulate REC viability due to radiation, chemotherapy or highglucose, and therefore prevent, treat or cure RNV-caused blindness.

SUMMARY OF THE INVENTION

The invention relates to a method for regulating REC viability in amammal by administering to the mammal a therapeutically effective amountof a quinic acid analogs (QAAs) having a structure as in Formula I.

In Formula I, the ring may be singly, doubly, or completely saturated;R¹ and R² are each independently H, straight or branched alkyl, aryl,benzyl, arylalkyl, or heterocyclic amine; R³ may be present or absentand, if present, may be H, hydroxyl, ether, alkoxy, or aryloxy; and R⁴,R⁵, and R⁶ are each independently H, hydroxyl, or alkoxy.

In some embodiments, R¹ and R² of Formula I form a piperidine ring withnitrogen (N). In other embodiments, when one of R¹ or R² of Formula I ishydrogen, the other of R¹ or R² is alkyl. In one instance of QAA, thealkyl is —C₃H₇ and each of R³-R⁶ is hydroxyl.

In some embodiments, the method may be used to prevent, treat or cureradiation retinopathy due to exposure of the mammal's RECs to radiation.For example, the radiation such as (gamma) γ radiation may be used totreat an intraocular tumor.

In other embodiments, the method may be used to prevent, treat or cureeye complications caused by chemotherapy in treating retinoblastoma. Thechemotherapy may be super-selective intra-ophthalmic arterychemotherapy. In one instance, the chemotherapy may employ the substancemelphalan.

In still other embodiments, the method may be used to prevent, treat orcure diabetic retinopathy. Diabetic retinopathy may be due to long timeexposure of the mammal's RECs to high glucose levels. In some instances,the mammal may be a human.

In still other embodiments, the method may be used to regulate RECviability for purposes of promoting or maintaining REC viability. Inother embodiments, the purpose is to reduce REC death. In furtherembodiments, the purpose is to prevent or reduce retinalneovascularization.

In some embodiments, the method is used in combination with one or moreexisting treatment methods for radiation retinopathy, diabeticretinopathy, or mitigating side effects on RECs in treatingretinoblastoma. In some instances, the quinic acid analog used in themethod is formulated in nanoemulsion and may be delivered as aneye-drop.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a model where irradiation of the eye triggers leukocyteadhesion, blockage, and nutrient/oxygen deprivation of retinalvasculature. Resultant hypoxia leads to dysfunctional retinalneovascularization and vision loss.

FIGS. 2A and 2B show graphs and images depicting the results of humanREC adhesion assays. In FIG. 2A, the x-axis shows three groups ofexperiments: control group referring to human RECs without irradiationor KZ-41 treatment; IR group referring to human RECs treated withirradiation; and IR+KZ-41 group referring to human RECs treated withboth irradiation and KZ-41. The y-axis shows the percentage of meanfluorescence values for each group. FIG. 2B shows a graph depicting theresults of monocyte adhesion assays in the upper panel and images ofcells in plates in the lower panel. In upper panel graph, the x-axisshows three groups of experiments: control group referring to human RECswithout irradiation or KZ-41 treatment; irradiation group referring tohuman RECs treated with irradiation; and IR+KZ-41 group referring tohuman RECs treated with both irradiation and KZ-41. The y-axis showsnumber of monocyte cells per field for each group. The images in thelower panel shows the monocyte cells adhered to the human RECs in eachof the three groups: A for the Control group; B for irradiation group;and C for the IR+KZ-41 group.

FIGS. 3A and 3B show images and graphs depicting the results of assayingICAM-1 expression upon KZ-41 treatment. FIG. 3A shows images taken withconfocal microscopy of flow-chamber slides. In FIG. 3A, the top panel(A, D and G), the middle panel (B, E and H), and the lower panel (C, Fand I) represent overlay images of DAPI, P-Selectin and ICAM-1,respectively. The columns A-C, D-F, G-I represent the three treatmentgroups: control group referring to human RECs without irradiation orKZ-41 treatment; IR group referring to human RECs treated withirradiation; and IR+KZ-41 group referring to human RECs treated withboth irradiation and KZ-41, respectively. FIG. 3B shows an imagedepicting the immunoblotting of ICAM-1 extracted human RECs from thesame three treatment groups as in FIG. 3A (lower panel) and a graphdepicting the quantity of ICAM-1 in the immunoblottings (upper panel).In the graph, the x-axis shows the three treatment groups. The y-axisshows the percentage of the amount of IAM-1 over the amount of thecontrol group.

FIGS. 4A and 4B show images and graphs depicting the results ofp38^(MAPK) assays in human RECs. In FIG. 4A, the lower panel showsimages of immunoblotting following high-dose irradiation (30 Gy) ofhuman RECs at different time points. The upper panel shows a graphdepicting the quantities of phosphorylated p38^(MAPK) at different timepoints. The x-axis has each of the time point. The y-axis is thepercentage of the quantitative ratio of phosphorylated p38^(MAPK) overtotal p38^(MAPK) at each time point in comparison to the quantitativeratio phosphorylated p38^(MAPK) over total p38^(MAPK) at time pointzero. In FIG. 4B, the lower panel shows images of immunoblotting inthree groups of human RECs: control group referring to human RECswithout irradiation or KZ-41 treatment; irradiation group referring tohuman RECs treated with irradiation (30 Gy) for 4 hours; and IR+KZ-41group referring to human RECs treated with both irradiation (30 Gy) for4 hours and KZ-41, respectively. The upper panel shows a graph depictingthe quantities of phosphorylated P38 in the three groups of human RECs.The x-axis shows each of the three treatment groups. The y-axis showsthe percentage of the quantitative ratio of phosphorylated P38 overtotal P38 in each group in comparison to the quantitative ratiophosphorylated p38^(MAPK) over total p38^(MAPK) in the control group. Inboth FIGS. 4A and 4B, P-p38^(MAPK) refers to phosphorylated p38^(MAPK)protein; Total p38^(MAPK) refers to both phosphorylated andunphosphorylated p38^(MAPK) protein; and α-Tubulin refers to thenormalization control protein α-Tubulin.

FIGS. 5A, 5B, 5C, 5D, and 5E show images and graphs depicting theresults of assays of p53 protein and/or phosphorylated p53. FIGS. 5A, 5Band 5C show graphs depicting the expression of p53 with phosphorylatedserine at positions 15, 33, 37, respectively. The x-axis shows the threetreatment groups: control group referring to human RECs withoutirradiation or KZ-41 treatment; IR group referring to human RECs treatedwith irradiation; and IR+KZ-41 group referring to human RECs treatedwith both irradiation and KZ-41, respectively. The x-axis shows thepercentage of the quantitative ratio of phosphorylated p53 over totalp53 in each group in comparison to the quantitative ratio ofphosphorylated p53 over total p53 in the control group. NS means thedifference is not significant. The symbol * means the difference issignificantly different. The ratio of the amount of phosphorylation p53to the amount of total p53 in KZ-41-treated RECs shows no significantreduction (P>0.05). FIG. 5D shows the graph depicting the total p53protein in the three groups of human RECs (upper panel) and an image ofthe immunoblotting depicting the expression of the three phosphorylatedp53, total p53 and the GAPDH control protein, in the three groups ofhuman RECs (lower panel). In the upper panel graph, the x-axis shows thethree groups of human RECs. The y-axis shows the percentage of thequantitative ratio of total p53 over GAPDH in each group normalized tothe quantitative ratio of total p53 over GAPDH in the control group.When total p53 protein was normalized to GAPDH, there was a significantreduction in KZ-41 treated RECs (**P<0.05). FIG. 5E shows a graphdepicting the total p53 protein in four groups of human RECs (upperpanel) and an image of the immunoblotting depicting the expression oftotal p53 and the GAPDH control protein, in the four groups of humanRECs (lower panel). In the upper panel graph, the x-axis shows the fourgroups of human RECs (from left to right): the first group referring tohuman RECs without NSC652287 (RITA), KZ-41, and SB 202190 (p38^(MAPK)inhibitor) treatments; the second group referring to human RECs treatedwith NSC652287 (RITA) only; the third group referring to human RECstreated with both NSC652287 (RITA) and KZ-41; the fourth group referringto human RECs treated with both NSC652287 (RITA) and SB 202190. They-axis shows the percentage of the quantitative ratio of total p53 overGAPDH in each group normalized to the quantitative ratio of total p53over GAPDH in the first group.

FIGS. 6A, 6B and 6C show graphs and images depicting the results ofassays of cleaved caspase-3 in three groups of human RECs: control groupreferring to human RECs without irradiation or KZ-41 treatment; IR groupreferring to human RECs treated with irradiation (30 Gy) for 24 hours;and IR+KZ-41 group referring to human RECs treated with both irradiation(30 Gy) for 24 hours and KZ-41, respectively. FIG. 6A shows the graphdepicting the amount of cleaved caspase-3 in the three groups of cellsby Sandwich ELISA. FIG. 6B shows images of in-cell Western results inthe three groups of cells. FIG. 6C shows the graph depicting the cleavedcaspase-3 protein amounts detected in the in-cell Western as shown inFIG. 6B. In both FIG. 6A and FIG. 6C, the x-axis shows the three groupsof cells and the y-axis shows the percentage of the amount of cleavedcaspase-3 in each group over the amount of cleaved caspase-3 in thecontrol group.

FIGS. 7A, 7B and 7C show graphs and images depicting the proliferativeand migratory phenotype of human RECs in different treatment groups.FIG. 7A shows a graph depicting the amount of VEGF protein in the humanREC culture medium in two groups: control group referring to cellswithout irradiation treatment and the IR-24 h group referring to cellstreated with irradiation (¹³⁷Cs 30 Gy, for 24 hours). The x-axis showsthe two groups. The y-axis shows the amount of VEGF in the medium(pg/mL). The symbol * in FIG. 7A means P<0.001. FIG. 7B shows an imageof Western blotting (upper panel and a graph depicting the results ofthe above Western blotting (lower panel) in four groups of human RECs:control referring to cells without irradiation, KZ-41 or SB202190treatments; IR-24 h group referring to cells treated with irradiation(¹³⁷Cs 30 Gy, for 24 hours); IR+KZ-41 referring to cells treated withirradiation (¹³⁷Cs 30 Gy, for 24 hours) and KZ-41; and IR+SB202190referring to cells treated with irradiation (¹³⁷Cs 30 Gy, for 24 hours)and SB202190. In the upper panel Western blotting image, p(Y118) refersto phosphorylated paxillin (Y118); paxillin refers to total paxillinincluding both phosphorylated and unphosphorylated paxillin; and GAPDHrefers to the control protein GAPDH. In the lower panel graph, thex-axis shows the four groups of human RECs and the y-axis shows thepercentage of the amount of ratio of phosphorylated paxillin to totalpaxillin in each group in comparison to the ratio of phosphorylatedpaxillin to total paxillin in the control group. In FIG. 7B, thesymbol * means P<0.01 and the symbol # means P<0.05. FIG. 7C shows agraph depicting the results of REC proliferation assays in threetreatment groups: vehicle control referring to cells without anyirradiation or KZ-41 treatment; irradiation referring to cells withirradiation treatment; and IR+KZ-41 (10 μM) referring to cells treatedwith irradiation and KZ-41 with a concentration of 10 μM. In FIG. 7C,the symbol * means P<0.05 and the symbol ** means P<0.05.

FIGS. 8A, 8B and 8C depict composition and characteristics of ocularnanoemulsion used for KZ-41 delivery. FIG. 8A shows the nanoemulsioncomposition consisting of Capryol 90 (7.5% v/v), Triacetin (7.5% v/v),Tween-20 (17.5% v/v) and Transcutol P (17.5% v/v) generated viahomogenization and water titration methods (left panel) and an image ofthe solution contained in a tube (right panel). FIG. 8B shows a graphdepicting the size of the nanoemulsion particles at day zero. After 60days at room temperature, the average particle size had increased to 75nm as shown in FIG. 8C. FIG. 8D shows a table depicting physicochemicalproperties of the nanoemulsion preparation: a viscosity of 17 mPa·s,average particle size of 60 nm at day zero, day 7 or after 3 cycles offreeze-thaw, and pH of 6.5.

FIG. 9 shows graphs and tables depicting results of ocularpharmacokinetic analysis after 100 mg/kg KZ-41 was administered by theophthalmic nanoemulsion method in mice. FIG. 9 shows a graph depictingthe concentration vs. time profile of KZ-41 in both ocular tissue andplasma. The ocular and plasma pharmacokinetic data are listed in thebottom tables. The time points are 0.08 hour, 0.25 hour, 0.50 hour, 1hour, 4 hour, 8 hour and 24 hour. SD means standard deviation. BLOQmeans below the limit of quantification.

FIGS. 10A and 10B show images and graphs depicting vascularization infour different treatment groups (A, B, C and D): normoxia referring tomice without treatments; OIR referring to mice treated with ocularirradiation; OIR+V referring to mice treated with ocular irradiation andvehicle ocular nanoemulsion without KZ-41; and OIR+KZ-41 referring tomice treated with ocular irradiation and ocular nanoemulsion with KZ-41,respectively. FIG. 10A shows images of avascular areas in the fourgroups of mice (upper panel) and a chart depicting the percentage ofavascular areas over total retinal vasculature in the four groups ofmice (lower panel). FIG. 10B shows images of neovascular areas in thefour groups of mice (upper panel) and the percentage of neovascularareas over total retinal vasculature in the four groups of mice (lowerpanel). In each group, 5 mice were tested. The percentage data representmean values (±standard deviations).

FIG. 11 shows a graph depicting the expression of cleaved caspase-3 indifferent group of human RECs: NG referring to RECs cultured in normalglucose levels at a concentration of 5 mM and treated with normal salinefor two hours; Mannitol referring to RECs cultured 25 mM mannitol andtreated with normal saline for two hours; HG referring to RECs culturedin high glucose levels at a concentration of 25 mM and treated withnormal saline for two hours; NG+KZ41 referring to RECs cultured innormal glucose levels (5 mM) and treated with KZ-41 at 10 μM for twohours; M+KZ-41 referring to RECs cultured in mannitol (25 mM) andtreated with KZ-41 at 10 μM for two hours; and HG+KZ41 referring to RECscultured in high glucose levels (25 mM) and treated with KZ-41 at 10 μMfor two hours. The amounts of cleaved capase-3 are represented inoptical density (O.D. at 450 nm) with mean value±standard deviation. Ineach group, 6 independent assays were conducted. The symbol * meansP<0.05 versus NG. The symbol # means P<0.05 versus HG (Student'st-test). Cleaved caspase-3 levels were measured by PathScan ELISA usingan antibody specific for cleaved caspase-3 (Asp175) Rabbit mAb.

FIGS. 12A and 12B show images and graphs depicting the expression ofphosphorylated Akt protein in different treatment groups. FIG. 12A showsa Western Blotting image depicting the expression of phosphorylated Aktprotein, total Akt protein, and control GAPDH protein in RECs culturedin high glucose at different times and a graph depicting the ratio ofphosphorylated Akt protein versus total Akt protein at different timepoints. At each time point, three independent samples were assayed. FIG.12B shows a Western Blotting image depicting the expression ofphosphorylated Akt protein and total Akt protein in six groups of RECcells as described in FIG. 11. In each group, 3 cell cultures wereperformed and assayed. The symbol * means P<0.05 versus NG. The symbol #means P<0.05 versus HG. All data are presented as mean values±standarddeviation.

FIGS. 13A and 13B show Western Blotting images and graphs depicting theexpression of phosphorylated Akt/total Akt and phosphorylated p85/totalp85 in different treatment groups of RECs. FIG. 13A shows an image ofWestern Blotting depicting the expression of phosphorylated Akt, totalAkt and control GAPDH protein in REC cells cultured in high glucose (25mM, HG) pretreated with or without LY294002 (10 μM) for 3 h, thentreated with or without KZ-41 (10 μM) for 2 hr. FIG. 13B shows a WesternBlotting image depicting the expression of phosphorylated p85, total p85and control GAPDH in RECs cultured in normal (5 mM) or high glucose (25mM) and then treated with or without KZ-41 (10 μM) for 2 hours (upperpanel), and a graph depicting the results in the Western Blotting image(lower panel). The x-axis shows the four groups of RECs. The y-axisshows the ratio of phosphorylated p85 to total p85 in each group. Thesymbol * means P<0.05 versus NG. The symbol # means P<0.05 versus HG. Ineach group, 3 cell cultures were performed and assayed. All data arerepresented as mean values±standard deviations.

FIG. 14 shows a Western Blotting image depicting the expression of IRS-1and GAPDH proteins in five groups of REC cells (upper panel) and a graphdepicting the results in the Western Blotting image (lower panel). Thefive groups of RECs are RECs cultured in normal glucose (5 mM, NG), highglucose (25 mM, HG), Mannitol (25 mM, M), normal glucose with KZ-41treatment (10 μM for 2 hr) and high glucose with KZ-41 treatment (10 μMfor 2 hr). The symbol * means P<0.05 versus NG. The symbol # meansP<0.05 versus HG. In each group, 3 cell cultures were performed andassayed. All data are represented as mean values±standard deviations.

FIG. 15A shows a Western blotting image depicting the expression ofphosphorylated IGF-1R^(1135/1136), total IGF-1R, and GAPDH proteins indifferent REC groups. REC cells cultures in normal glucose (5 mM, NG),high glucose (25 mM, HG), Mannitol (25 mM, M), normal glucose with KZ-41treatment (10 μM for 2 hr), and high glucose with KZ-41 treatment (10 μMfor 2 hr). FIG. 15B shows a graph depicting the ratio of phosphorylatedIGF-1R^(1135/1136) to total IGF-1R in each of the five groups. FIG. 15Cshows a graph depicting the ratio of total IGF-1R to GAPDH in each ofthe five groups. FIG. 15D shows a graph depicting the ratio ofphosphorylated IGF-1R^(1135/1136) to GAPDH in each of the five groups.The symbol * means P<0.05 versus NG. The symbol # means P<0.05 versusHG. In each group, 3 cell cultures were performed and assayed. All dataare represented as mean values±standard deviations.

FIG. 16A shows a graph depicting cell death ELISA (Roche) results ofRECs treated with nothing (control), melphalan (4 μg/mL) andmelphalan+various doses of KZ-41. The symbol * means P<0.05 vs. control.The symbol # means P<0.05 vs. melphalan only. In each group, 4 cellcultures were performed and assayed. FIG. 16B shows a graph depictingcell death ELISA (Roche) result of Y79 retinoblastoma cells with notreatment (control), 0.8 μg/mL, 1 μg/mL or 4 μg/mL melphalan todemonstrate that melphalan best induces apoptosis at 4 μg/mL. FIG. 16Cshows a graph depicting cell death ELISA (Roche) results of Y79retinoblastoma cells treated with melphalan at 4 μg/mL alone or incombination with KZ-41. KZ-41 did not inhibit melphalan-inducedapoptosis of Y79 cells. The symbol * means P<0.05 vs. control. Thesymbol # means P<0.05 vs. melphalan only. In each group, 4 cell cultureswere performed and assayed. All data are represented as meanvalues±standard error of the mean.

FIG. 17A shows a graph depicting the ICAM-1 ELISA results for RECstreated with nothing, melphalan only, or melphalan+KZ-41 (10 uM). They-axis shows ICAM-1 concentration in ng/mL. FIG. 17B shows a graphdepicting the ICAM-1 Western blot results of RECs untreated, melphalanonly, KZ-41 or Melphalan+KZ-41. The y-axis shows the intensity ratio ofICAM-1 to Actin. In both ELISA and Western blot results, KZ-41 reducedmelphalan-induced ICAM-1 levels in RECs. The symbol * means P<0.05 vs.control. The symbol # means P<0.05 vs. melphalan only. In each group, 4cell cultures were performed and assayed. All data are represented asmean values±standard error of the mean.

FIGS. 18A and 18B show graphs depicting the ICAM-1 ELISA results of RECstreated with (from left to right) sc siRNA, TNFα siRNA (FIG. 18A) oretanercept (FIG. 18B) and melphalan. Neither TNFα siRNA nor etanerceptreduced ICAM-1 levels after melphalan treatment. The y-axis shows ICAM-1concentration in ng/mL. FIG. 18C shows a graph depicting cell deathELISA (Roche) results of RECs treated with etanercept and/or melphalan.The y-axis shows DNA fragmentation in O.D. 405 nm. While etanercept didreduce melphalan-induced REC apoptosis, it did not reach statisticalsignificance. The symbol * means P<0.05 vs. control. The symbol # meansP<0.05 vs. melphalan only. In each group, 4 cell cultures were performedand assayed. All data are represented as mean values±standard error ofthe mean.

FIG. 19A shows images and a graph depicting Western Blot results for theratio of Phospho-NF-κB to total NF-κB after melphalan treatment. FIG.19B shows images and a graph depicting Western blot results for ratio ofPhospho-NF-κB to total NF-κB after melphalan and KZ-41 treatments. FIG.19C shows images and a graph depicting a control experiment todemonstrate that NF-κB siRNA was able to significantly reduce NF-κBprotein levels. FIG. 19D shows images and a graph depicting ICAM-1 ELISAresults after melphalan and NF-κB siRNA treatment. The symbol * meansP<0.05 vs. control. The symbol # means P<0.05 vs. melphalan only. Ineach group, 4 cell cultures were performed and assayed. All data arerepresented as mean values±standard error of the mean.

FIG. 20A shows images and a graph depicting the expression ofPhospho-P38^(MAPK) and total P38^(MAPK) in RECs with or withouttreatment with KZ-41. FIG. 20B shows a graph depicting the ICAM-1 ELISAresults in RECs treated with various combinations of melphalan, KZ-41,the P38^(MAPK) inhibitor SB202190. The symbol * means P<0.05 vs.control. The symbol # means P<0.05 vs. Melphalan. The symbol $ meansP<0.05 vs. KZ-41. The symbol & means P<0.05 vs. KZ-41+Melphalan. In eachgroup, 4 cell cultures were performed and assayed. All data arerepresented as mean values±standard error of the mean.

FIG. 21A shows a graph depicting ICAM-1 levels in RECs treated withICAM-1 siRNA and/or melphalan. The Y-axis shows the ICAM-1concentrations at ng/ml. FIG. 21B shows a graph depicting cell deathELISA results of RECs treated with ICAM-1 siRNA and/or melphalan. FIG.21C shows a graph depicting cell death ELISA results of RECs treatedwith various combinations of melphalan, KZ-41, and SB 202190. FIG. 21Dshows a graph depicting cell death ELISA results of RECs treated withNF-κB siRNA and or melphalan. The symbol * means P<0.05 vs. control. Thesymbol # means P<0.05 vs. Melphalan. The symbol $ means P<0.05 vs.KZ-41. The symbol & means P<0.05 vs. KZ-41+Melphalan. In each group, 4cell cultures were performed and assayed. All data are represented asmean values±standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is partly based on the surprising discovery that anew class of quinic acid analogs (QAAs) can regulate REC viability. TheQAAs therefore can be used to therapeutically prevent, treat, or curepathological conditions associated with RECs of various etiologies,including but not limited to, radiation, chemotherapy or diabetes. Onesuch pathological condition, for example, is retinalneovascularization-caused blindness. The QAAs have been previouslydescribed in U.S. Pat. No. 8,115,031 and U.S. Patent ApplicationPublication No. 2012/0283331, both of which are incorporated herein byreference in their entireties.

As such, in one aspect, the present invention is directed to a methodfor regulating REC viability in a mammal by administering to the mammala therapeutically effective amount of a QAA compound having a structureas in Formula I.

In Formula I, the ring may be singly, doubly, or completely saturated;R¹ and R² are each independently H, straight or branched alkyl, aryl,benzyl, arylalkyl, or heterocyclic amine; R³ may be present or absentand, if present, may be H, hydroxyl, ether, alkoxy, or aryloxy; and R⁴,R⁵, and R⁶ are each independently H, hydroxyl, or alkoxy.

In some embodiments, R¹ and R² in Formula I of the QAA compound form apiperidine ring with Nitrogen (N). In other embodiments, when one of R¹or R² of Formula I of the QAA compound is hydrogen, the other of R¹ orR² is alkyl. In one instance of the latter embodiments, the compound isa QAA herein named “KZ-41” where the alkyl is —C₃H₇ and each of R³-R⁶ ishydroxyl.

In other embodiments, R³-R⁶ in Formula I may be an antioxidant with anester bond in between the antioxidant and the ring. In some instances,only one of R³-R⁶ is an antioxidant. In other instances, any two ofR³-R⁶ are antioxidants. In still other instances, any three of R³-R⁶ areantioxidants. In still other instances, all R³-R⁶ are antioxidants.Antioxidants may a naturally occurring antioxidant, including, but notlimited to, caffeic acid, ferulic acid, and sinapic acid. In oneembodiment, the antioxidant is a caffeic acid which is connected throughan ester bond to the ring at anyone of the positions R³-R⁶.

QAAs may be synthesized by methods as previously described in U.S. Pat.No. 8,115,031. Alternatively, QAAs may be isolated from a variety ofplant extracts, such as Cat's Claw extract. Methods of isolation areknown in the art (Akesson et al., 2005; Sheng et al., 2005).

In some embodiments, the mammal may be any mammal that needs treatmentfor pathological conditions associated with retinal endothelial cells.In some preferred embodiments, the mammal is a human and the method ofadministering QAAs is used to promote viability of RECs in anticipationof or post-ocular insults.

In some embodiments, QAAs may be administered in a pharmaceuticalcomposition containing the compound in combination with other chemicalcomponents such as physiologically suitable carriers and excipients inorder to facilitate administration of QAAs to a target site. Suchpharmaceutical compositions can be prepared by methods and containexcipients which are well known in the art. Such methods and ingredientsmay be found in Remington's Pharmaceutical Sciences (Alfonso Gennaro etal., eds., Lippincott, Williams & Wilkins, Baltimore, Md., 20th ed.,2000).

For example, a pharmaceutically acceptable carrier may be a carrier, anadjuvant or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. An excipient may be an inert substance addedto a pharmaceutical composition to further facilitate administration ofan active ingredient. Examples, without limitation, of excipientsinclude calcium carbonate, calcium phosphate, various sugars and typesof starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

Proper formulation of QAA is dependent upon the route of administrationchosen. For example, as described in Example 1, QAA may be formulated innanoemulsion so that QAA may be delivered to the retinal cells topicallyas an eye-drop.

Suitable routes of systematic administration of QAA may, for example,include oral, rectal, transmucosal, especially transnasal, intestinal orparenteral delivery, including intramuscular, subcutaneous andintramedullary injections as well as intrathecal, directintraventricular, intracardiac, e.g., into the right or left ventricularcavity, into the common coronary artery, intravenous, intraperitoneal,intranasal, or intraocular injections. Alternately, one may administerQAA in a local rather than systemic manner, for example, via injectionof the pharmaceutical composition directly into a tissue region of apatient, for example, the eye.

A therapeutically effective amount is an amount of QAA necessary toachieve the desired endpoint. One desired endpoint, for example, is toprevent the occurrence of REC death following radiation therapy ofocular tumor, chemotherapy of retinoblastoma, or exposure of eye to highglucose. Additional desired endpoints may, for example, includedecreased expression of proteins such as cleaved caspase-3 and ICAM-1that are associated with apoptosis of RECs. Still additional desiredendpoints may for example, include phenotypes such as decreasedavascular area or neovascular areas, or decreased apoptosis of RECs.

Assessment of a therapeutically effective amount is well within theskill of one in the medical and pharmaceutical arts, given thedisclosure herein. For example, the U.S. Department of Health and HumanServices Food and Drug Administration Center for Drug Evaluation andResearch (CDER) has established guidance for estimating dosages(Guidance for Industry: Estimating the Maximum Safe Starting Dose inInitial Clinical Trials for Therapeutics in Adult Healthy Volunteers,July 2005).

Therapeutically effective doses may be achieved via administration of asingle dose, but may also be achieved via administration of more thanone dose, such as an initial dose in combination with one or moreadditional doses which may be provided within a specific timeframe, forexample, such as within about 12 to about 72 hours after the initialdose.

QAA may, if desired, be presented in a pack or dispenser device, such asan FDA approved kit, which may contain one or more unit dosage formscontaining the active ingredient. The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accommodated by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions or human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert.

In one embodiment, QAA is used to prevent, treat or cure radiationretinopathy. Radiation retinopathy (RR) is a chronic degenerativedisease that leads to significant visual impairment (Giuliari et al.,2011). RR results from exposure of the eye to various directedradiotherapy interventions (e.g., external beam, plaque brachytherapy,and gamma knife) (Egger et al., 2001; Finger et al., 2009; Haas et al.,2002; Krema et al., 2009; Witt, 2003). Radiotherapy is increasingly usedto treat intraocular tumors since it provides equivalent or enhancedsurvival compared to enucleation (eye removal) (Diener-West et al.,2001; Jampol et al., 2002; Phillpotts et al., 1995). The incidence ofproliferative (i.e., neovascular) RR was recently reported as 7% at tenand fifteen years in 3,841 eyes treated with plaque radiotherapy foruveal melanoma while overall estimates of RR incidence are as high as20% (Bianciotto et al., 2010; Gunduz et al., 1999). Patients thatconsequently undergo secondary enucleations, in spite of successfulradiotherapy, do so because of extensive retinal vascular damage. Severepain and/or vision loss associated with lesions, inflammation,thrombosis, and neovascularization were present in a majority of thesecases (Avery et al., 2008).

Radiation-induced damage to the vascularized posterior retinal segmentsof the eye triggers an exuberant pro-inflammatory response resulting inleukocyte adhesion/stasis, vessel occlusion, retinal endothelial cell(REC) death, and subsequent hypoxia (Cai et al., 2004; Johnston et al.,2003; Olthof et al., 2001). The ischemic state within the retina triggergrowth-factor mediated neovascularization, secondary to the initialradiation injury. Hallmark vision-threatening cytopathological featuresof retinal inflammation are vascular leakage and capillarynon-perfusion, contributed in large part to the accumulation of immunecells in the damaged areas (Miyamoto et al., 1999). Thus, this breakdownand compensatory neovascularization within the highly vascularizedretina leads to blindness. The term “radiation” means “ionizingradiation.” examples of ionizing radiation including, but not beinglimited to, gamma-radiation sources and x-rays.

In another embodiment, QAA is used to prevent, treat or cure sideeffects caused by chemotherapy of retinoblastoma. Retinoblastoma is themost common primary intraocular malignancy in children. With treatment,greater than 90% of patients in developed countries live. However, toimprove the quality of life of these cancer survivors, newer treatmentsusing localized chemotherapy have been developed in hopes of bettersalvaging eyes and vision. Selective ophthalmic artery chemotherapy wasfirst developed by Kaneko in the 1980's using a balloon occlusion of thecarotid artery to selectively deliver chemotherapy to the ophthalmicartery (Pham et al., 2012; Yamane et al., 2004). Subsequently, Abramsonmodified Kaneko's approach by directly delivering melphalan, a nitrogenmustard, into the ophthalmic artery under manual control without balloonocclusion, a technique termed super-selective intra-ophthalmic arterychemotherapy (SSIOAC) (Abramson et al., 2008; Gobin et al., 2011). WhileSSIOAC may be more effective than systemic chemotherapy for specificcases, recent data indicates that a number of significant side effectsto the retinal and choroidal vasculature can occur following SSIOAC withmelphalan (Munier et al., 2011; Shields et al., 2011; Wilson et al.,2011). For example, melphalan SSIOAC administered to non-human primates(NHPs), in the same manner as given to children, produced retinalvascular inflammation, manifested as increased leukocyte adhesion, andocclusion. (Steinle et al., 2012). As such, in some embodiments, QAA isadministered to prevent, treat, or cure the side effects caused bychemotherapy of retinoblastoma. These side effects include retinalvascular inflammation, which can lead to choroidal vasculature,neovasculature, retinal endothelial cell death and eventually blindness.For instance, as shown in Example 3, KZ-41 may be administered toprotect retinal endothelial cells against melphalan-induced damagewithout affecting the apoptosis of melphalan-treated humanretinoblastoma cells.

In some embodiments, QAA administrations may start prior to radiationtherapy or chemotherapy, and continue during radiation therapy orchemotherapy, and then after radiation therapy or chemotherapy until therisk of radiation therapy or chemotherapy-induced damage is minimal. Inother embodiments, QAA administrations may start in conjunction withradiation therapy or chemotherapy, and then continue after radiationtherapy or chemotherapy. In still other embodiments, QAA administrationsmay start after radiation therapy or chemotherapy. Depending whetherpathological conditions have occurred when QAA is administered, QAA isused to prevent, treat or cure the pathological conditions caused byradiation therapy or chemotherapy.

In yet another embodiment, QAA is used to prevent, treat or curediabetic retinopathy. Diabetic retinopathy (DR), one of the mostfrequently occurring microvascular complications of diabetes, is aleading cause of visual loss in patients aged 20-74 years (Bassoli etal., 2008; Marshall and Flyvbjerg, 2006). Numerous investigations havesuggested that the pathogenesis of diabetic retinopathy includesglucose-mediated anatomic changes in retinal vessels (Engerman, 1989).Blood retinal barrier issued vascular leakage and pre-retinalneovascularization are key clinical features of DR. Hyperglycemiatriggers caspase-dependent apoptosis in retinal endothelial cells, whichis followed by a compensatory angiogenic response to replace lost cells(Geraldes et al., 2009). The cycle of accelerated REC death and renewalis thought to contribute to vascular architectural changes and, uponexhaustion of the replicative life. Laser photocoagulation to reduceneovascularization and macular edema has been the mainstay of treatmentfor proliferative diabetic retinopathy (PDR) more than 25 years.However, it is a destructive therapy, with adverse side effects such asloss of peripheral visual filed and night vision as well as exacerbationof macular edema and subsequent reduction of central vision (Shimura etal., 2003). Therefore, it is imperative to develop new identification,treatment options to treat retinopathy in the earliest stages.

As such, in some embodiments, QAA is administered to prevent, treat orcure the vascular complications and subsequent visual loss caused byexposure to high glucose. In some instances, QAA administrations maystart before any symptoms of diabetic retinopathy occur so that theoccurrence of diabetic retinopathy may be postponed, delayed, orprevented. In other instances, QAA administrations may start aftersymptoms of diabetic retinopathy occur so that diabetic retinopathy maybe prohibited from further exacerbation or the exacerbation may bedelayed or postponed.

The common pathological conditions caused by radiation therapy, highglucose exposure or chemotherapy include retinal endothelial cell death(“REC death”) which in turn results in retinal neovascularization in theretina. As such in some embodiments, QAA is administered to regulate theviability of retinal endothelial cells so that REC death caused byvarious ocular insults may be prevented or reduced. In otherembodiments, QAA is administered to regulate the viability of retinalendothelial cells so that retinal neovascularization caused by variousocular insults may be prevented or reduced.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

It should be understood that this invention is not limited to theparticular methodologies, protocols and reagents, described herein,which may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention.

Examples of the disclosed subject matter are set forth below. Otherfeatures, objects, and advantages of the disclosed subject matter willbe apparent from the detailed description, figures, examples and claims.Methods and materials substantially similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentlydisclosed subject matter. Exemplary methods and materials are nowdescribed as follows.

Example 1 KZ-41 could Regulate Retinal Endothelial Cell Viability inConnection with Radiation Retinopathy

In this Example 1, we demonstrated that KZ-41 could be used to regulateretinal endothelial cell viability in both in vitro and in vivo modelsof RR. We discovered that KZ-41 functioned by modulating theseinflammatory elements in primary RECs acutely exposed to ionizingradiation (IR). The attenuation of inflammatory responses triggered byhigh-dose irradiation by KZ-41 suggested that KZ-41 treatment can beused as a pre-exposure prophylactic measure to limit and/or preventpathological precursors of RR (i.e., retinal vascular inflammation).

The materials and methods we used in this example are as follows.

Reagents/Antibodies.

KZ-41 was synthesized in Dr. Duane Miller's laboratory and verified tobe >96% pure by nuclear magnetic resonance spectroscopy (Zeng et al.,2009). Calcein-AM was obtained from BD Biosciences (San Jose, Calif.).Conjugated ICAM-1 (sc-107 PCPC5) and P-selectin (sc-19672 FTIC)antibodies for confocal microscopy were purchased from Santa CruzBiotechnology (Santa Cruz, Calif.). DAPI nuclear stain was obtained fromPierce (Rockford, Ill.). Phosphorylated (Thr180/Tyr182) p38MAPK primaryantibodies were purchased from R&D Systems (Minneapolis, Minn.).Phosphorylated (Ser-15, -33, -37) and total p53, p38MAPK, unconjugatedICAM-1, phosphorylated (Tyr118) and total paxillin, and GAPDH primaryantibodies were acquired from Cell Signaling (Danvers, Mass.).Alpha-Tubulin primary antibody and secondary antibodies, IRDye 800CWgoat anti-rabbit and IRDye 680LT goat anti-mouse were purchased fromLI-COR Biotechnology (Lincoln, Nebr.). SB202190 and NSC652287,inhibitors of p38^(MAPK) and p53-MDM2, respectively were purchased fromTocris Bioscience (Bristol, UK).

Cell-Culture.

Primary human retinal microvascular endothelial cells (REC, Lot 181)were provided by Cell Systems Corporation (CSC, Kirkland, Wash.). Cellswere grown on gelatin-coated surfaces in M131 medium containingmicrovascular growth supplements (MVGS) (Invitrogen; Carlsbad, Calif.),gentamycin (10 mg/mL), and amphotericin B (0.25 mg/mL). Only primarycells within passage 6 were used. For immunoassays, RECs were platedinto six-well plates and cultured for two days. RECs were pretreatedwith KZ-41 (10 μM) for 12 hours and then exposed to gamma (γ)-rays (30Gy) using a Shepherd Mark I, model 68, 137Cs irradiator (J.L. Shepherd &Associates, San Fernando, Calif.) at a dose rate of approximately 3Gy/min. To investigate cell signaling through p38^(MAPK), SB202190 wasadded to culture medium 30 minutes prior to IR. U937 (humanmonocytic-like) cells (ATCC, Manassas, Va.) were cultured in RPMI 1640(Invitrogen) supplemented with 10% fetal bovine serum, penicillin (5000IU) and streptomycin (5 mg/mL). Cells to passage 10 were used foradhesion experiments.

Static Cell Adhesion.

Cellular adhesion under static conditions was assessed using amicroplate assay as previously described (Chang et al., 2010; Yu et al.,2007). Briefly, human primary RECs (105 cells/well) were seeded to96-well plates and cultured to a confluent monolayer. RECs were treatedwith either KZ-41 (10 μM) or vehicle (0.9% normal saline), irradiated at30 Gy and incubated for 24 hours at 37° C. Calcein-AM loaded U937 cellswere added to REC-containing wells and allowed to adhere for 30 minutes.Non-adherent cells were removed from wells by gentle washes and adhesionwas quantified using a fluorescence microplate reader(excitation/emission wavelengths of 485/535 ηm). Data represent meanfluorescence±standard deviation (SD) normalized to backgroundfluorescence.

Parallel-Plate Flow Chamber.

Cell adhesion under physiological fluid-shear was investigated using aparallel-plate flow chamber and continuous flow-loop (Cytodyne Inc., LaJolla, Calif.) at a shear stress of 2 dyne/cm2 (Lawrence et al., 1987;Steinle et al., 2012; Wagers et al., 1998). Shear stress within thechamber was determined using a constant fluid flow-rate calibrated byadjusting the height of the hydrostatic inlet and outlet ports of thefluid reservoir (Frangos et al., 1988; Wagers et al., 1998). The flowrate for the required shear stress was calculated using the followingequation: SS=6Qμ/bh2, where SS=shear stress (dyne/cm2), Q=flow rate(cm3/s), μ=fluid viscosity (dyne*s/cm2), b=chamber width (cm), h=chamberheight (cm). RECs were seeded onto gelatin-coated microscope slides(75×38 mm; Corning Inc., Corning, N.Y.) and grown to confluence. KZ-41(10 μM) or vehicle-treated RECs were irradiated at 30 Gy and incubatedfor 24 hours. Slides were then placed into the chamber and U937 (humanmonocytic) cells (2.5×10⁶ cells/mL) were perfused over the RECmonolayer. Interacting monocytes were monitored over two hours using atleast eight different fields of view and digitally recorded for off-lineanalysis. Phase contrast images of U937 cells adhering to human RECmonolayers were obtained using a Nikon Diaphot 300 phase-contrastmicroscope (Nikon, Melville, N.Y.) equipped with a Dage-MTI series 68camera (Dage-MTI, Michigan City, Ind.). High-resolution video and imageswere analyzed using Adobe Premier Pro CS5.5 (Adobe Systems, Inc., SanJose, Calif.). Firm adhesion was defined as interacting monocytesremaining stationary at each 30-minute increment (Alon et al., 1996;McCarty et al., 2000). After two hours, RECs were removed from the flowchamber and fixed in 4% formaldehyde for 15 minutes at room temperatureand washed three times with ice-cold phosphate-buffered saline (PBS).Data from three separate experiments represent mean adherent cells/fieldof view over 30 minute increments±SD.

Confocal Microscopy.

Non-specific blocking of proteins on cellular surface was done using 10%bovine serum albumin (BSA) containing blocking buffer for at least onehour at room temperature. Human anti-ICAM-1 and anti-P-selectinantibodies conjugated to PerCp-Cy5.5, FITC respectively were diluted inPBS (1:50) and incubated with the slide for one hour at room temperaturewith gentle rocking Slides were then washed twice with cold PBS andincubated with DAPI nuclear stain for 10 minutes. Cells were againwashed and mounting medium along with cover slips were added to slidesand sealed prior to imaging. A Zeiss LSM 710 system with Zen 2010 v.6.0software (Carl Zeiss Microscopy, LLC, Thornwood, N.Y.) was used inacquisition and analysis of RECs. Adobe Photoshop CS5 (V. 12.1; AdobeSystems, Inc.) was used to measure relative intensities of confocalimages. All images were normalized to cell number. Data represent meanintensity signals±SD.

Immunoblot (Western) Analysis.

Irradiated RECs with or without treatments of either KZ-41 and/orinhibitors of p38^(MAPK) were carried out at 30 Gy. For ICAM-1 proteinlevel analysis, REC lysates were collected 24 hours after IR. Forphosphorylation status of MAPK and p53 stress pathways, REC lysates werecollected 4 hours following exposure to IR. Unirradiated RECs were takenout of the incubator during irradiations for environmental controls.Cellular proteins were analyzed by Western blot after SDS-PAGE usinghuman specific primary antibodies. REC lysates were collected in RIPAlysis buffer (50 mM Tris.HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NonidetP-40, 0.1% SDS) with protease/phosphatase inhibitor (1×) cocktail(Roche; Indianapolis, Ind.). Lysates were kept on ice and centrifuged at10,000 g for 10 minutes and total protein was measured using BCA assay(Pierce, Rockford, Ill.). Protein samples were mixed with 4×LDS loadingbuffer with 2.5% 2-mercaptoethanol (Sigma), heated to 70° C. for 10minutes, and loaded on NuPAGE 4-12% Bis-Tris gel (Invitrogen).Immunoblotting was performed with nitrocellulose membranes (Bio-Rad) at170-mA start and 110-mA end at 25 V for 2 h in NuPAGE transfer buffer(Invitrogen) containing 20% methanol. Membranes were blocked usingOdyssey blocking buffer (LICOR) for 1 h at room temperature with gentlerocking Membranes were then incubated at 4° C. with specific primaryantibodies (1:1000) overnight. Cellular protein was normalized usingGAPDH (Cell Signaling) or α-Tubulin (LI-COR) [1:20,000]. Secondaryantibodies (IRDye 800CW goat anti-rabbit and IRDye 680LT goatanti-mouse) (LI-COR) [1:10,000] were incubated in the dark at roomtemperature for 45 minutes. Dual-channel infrared scan and quantitationof immunoblots were conducted using the Odyssey® Sa infrared imagingsystem with Image Studio (Ver. 3.1.4) (LI-COR).

Cleaved Caspase-3.

In-Cell Western blotting (ICW) was used to quantitatively measure thepresence of cleaved caspase-3 protein within intact cells. Briefly, RECswere seeded into a 96 well plate and cultured for one day. Cells wereirradiated at 30 Gy and returned to incubator for 24 hours. Cells werethen fixed with 4% paraformaldehyde for 15 minutes with gentle shakingand washed five times with PBS containing 0.1% Triton X-100 solution.Odyssey blocking buffer was added to each well and incubated at roomtemperature for one hour. Cleaved caspase-3 primary antibody (CellSignaling) was diluted in blocking buffer (1:50) and incubated at 4° C.overnight with gentle shaking Wells were then washed with 0.1% Tween-20in PBS solution five times. IRDye 800CW goat anti-rabbit secondaryantibody (1:1000) was added along with DRAQ5 (Biostatus, Leicestershire,UK) counterstain for cell-number normalization. Cells were washed fivemore times with washing buffer and plates were imaged using the OdysseySa imaging system (LI-COR). Data represent background-normalized meanfluorescence intensity±SD (n=12/group). The Pathscan Cleaved Caspase-3sandwich ELISA kit (Cell Signaling) was performed to evaluate endogenouscaspase-3 activation 24 hours post-irradiation. Briefly, cells wereirradiated at 30 Gy and incubated for 24 hours in the presence orabsence of KZ-41. Lysates of equivalent total protein were used forELISA analysis. Assay was performed based on manufacturer's protocol.Data represent mean O.D.±SD.

VEGF-ELISA and REC Proliferation Assay.

ELISA assays were used to measure changes in protein levels of VEGF(Quantikine, Minneapolis, Minn.) Briefly, confluent RECs were irradiatedat 30 Gy and incubated at 37° C. for 24 hours; Medium was collected andanalyzed according to manufacturer's instructions. For all ELISAanalyses, equal protein amounts were loaded into each well, allowing forcomparisons using optical density (OD).

To evaluate KZ-41 modulation of irradiation-induced retinal endothelialcell proliferation 50,000 cells with or without KZ-41 (10 μM) wereplated into each well of a 96-well dish, irradiated at 30 Gy andincubated for 24 hours. Following treatment with KZ-41 or vehicle,cellular proliferation was determined using tetrazolium salt WST-1 and amicroplate reader (UQuant Reader; BioTek, Winooski, Vt.) according tothe assay manufacturer's instructions (Cell Proliferation Assay Kit, WSTdye, ELISA based; Millipore, Billierca, Mass.) at 450 nm. The absorbanceat 450 nm (recorded in Mean OD±SD) is directly correlated with cellularproliferation.

Nanoemulsion Formulation/Characterization and Ocular Pharmacokinetics.

Ocular nanoemulsion (NE) used for drug delivery was comprised of Capryol90 (7.5% v/v), Triacetin (7.5% v/v), Tween-20 (17.5% v/v) and TranscutolP (17.5% v/v) (Gattefossé Pharmaceuticals, Saint-Priest, France)generated via homogenization and water titration methods, as previouslydescribed (Ammar et al., 2009; Shafiq-un-Nabi et al., 2007).Characterization of the NE was done using an AR G2 Rheometer (TAInstruments, New Castle, Del.), Malvern Zetasizer (Malvern InstrumentsLtd, Malvern Worcestershire, UK), and Fisher Accumet Excel pH meter(Thermo Fisher Scientific, Waltham, Mass.). KZ-41 loaded NE wasdelivered to the eye for ocular PK studies at 100 mg/kg dose usingstandard pipetting techniques.

Male C57BL/6J (The Jackson Laboratory, Bar Harbor, Me.) mice weighing˜25 g were used for ocular administration of KZ-41 in NE. Animals (n=3)were sacrificed using cardiac puncture methods at specified time pointsbetween 5 minutes and 24 hours. Whole blood was separated viacentrifugation, and plasma was collected. Eyes were enucleated,irrigated with normal saline and homogenized to obtain compound fromocular chambers. Drug concentrations in both plasma and in the eye werestored at −80° C. until analysis. KZ-41 plasma and ocular samples wereprocessed and drug concentrations were determined using LC-MS/MS, aspreviously described (Ramagiri et al., 2009; Zeng et al., 2011). KZ-41plasma and ocular drug concentration-time data was analyzed usingnoncompartmental methods in WinNonlin 6.0 (Pharsight, Mountain View,Calif.).

Animals and Experimental Design of Oxygen-Induced Retinopathy (OIR)Murine Model.

C57BL/6J mice were used in these experiments. All animal experimentationwas performed under the guidelines of the Association for Research inVision and Ophthalmology for the humane use of animals in visionresearch, and in accordance with our established institutionalguidelines. Mouse pups were divided into four separate groups: 1)Untreated mice under ambient normal oxygen (normoxia) conditions(negative-control); 2) Untreated mice exposed to hyperoxia conditions(positive-control); 3) Nanoemulsion vehicle treated hyperoxia-exposedmice (vehicle-control); and 4) Hyperoxia-exposed mice treated withKZ-41-loaded nanoemulsion (compound-treated). A minimum number of 5animals were used for each experimental group.

Retinal neovascularization (RNV) was induced using a mouse model ofoxygen-induced retinopathy (OIR). Briefly, C57BL6/J mouse pups wereexposed to 75% oxygen at post-natal day 7 (P7) for 5 days and thenreturned to normal oxygen (P12). OIR mice received daily ocularadministration of either KZ-41 (100 mg/kg), vehicle (ocularnanoemulsion) or left untreated from P12 to P17. Normoxia (negativecontrols) mice were not manipulated during the study period. Retinasfrom all pups were removed, dissected, mounted and stained toinvestigate retinal angiogenesis as previously described (Arnold et al.,2012; Connor et al., 2009). For retinal whole mounts, enucleated eyesunderwent weak fixation (for ease of hyaloid vasculature removal) in 4%paraformaldehyde (PFA) in PBS for 1 h on ice and washed three times.Retinas were then isolated and mounted onto microscope slides. Wholeretinas were incubated overnight at 4° C. with isolectin B4-594 (AlexaFluor 594; Molecular Probes, Eugene, Oreg.). Isolectin-stained retinaswere then washed three times in 1×PBS, sealed on slides using ProlongGold (Invitrogen) and imaged.

Images were acquired using a Nikon Eclipse 80i confocal microscope andanalyzed with Nikon-NIS elements software (Nikon). Quantification ofavascular area (AV) and neovascularization (NV) in retinal whole mountswere performed in Adobe Photoshop (Adobe Systems, Inc.) as previouslydescribed (Arnold et al., 2012; Connor et al., 2009; Stahl et al.,2010). Briefly, the AV area was determined by the absence of isolectinstaining surrounding the optic disc. The area devoid of vascularizationwas characterized as a percentage of total retinal area (% AV).Quantification of NV was determined after threshold limits are setwithin software parameters. This technique ensured the quantification ofonly clusters and tufts of NV while excluding the normal vascularizedretina (less intense staining) Photoshop analysis tools were used tomanually outline NV formations and data was recorded as a percentage oftotal retinal area (% NV) (Arnold et al., 2012; Connor et al., 2009).

Statistical Analyses.

All data represented herein were performed in replicates of 3 or moreand presented as the mean±standard deviation (SD), unless otherwiseindicated. Analysis of variance (ANOVA) with Scheffe's post-hoc test wasused to compare mean values. Statistical significance was set at P<0.05.

We demonstrated herein that irradiation-induced monocyte adhesion toretinal endothelial cells in both static and fluid environments could beattenuated with the treatment of KZ-41. Here, we investigated the acuteeffects of a single total dose of approximately 30 Gy (˜3 Gy/min)delivered in vitro. The rapid induction of ICAM-1 in a variety ofendothelial cells is one of major inflammatory indicators of exposure tohigh-dose radiation (Gaber et al., 2003; Yuan et al., 2003). Using afluorescent based static-adhesion assay we determined that in responseto irradiation, primary human RECs could elicit an inflammatory responsevia monocyte adhesion. As shown in FIG. 2A, adhesion of U937 monocyticcells was enhanced two-fold in RECs 24 hours post irradiation (P<0.005)and RECs treated with KZ-41 immediately prior to radiation experienced asubstantial decrease in the number of adhering cells (P<0.005).

In blood flow, tethering and rolling are initiated by selectins (e.g.,L-, P- and E-selectin) which are rapidly presented to the cell surfacein response to inflammatory stimuli [37]. Consequently, selectins andother immunoglobin superfamily of cellular adhesion molecules (CAMs;e.g., ICAM, VCAM, PECAM, etc.) are able to perform similar adhesivefunctions in static environments where the lack of sheer stress promotesadhesive-like interactions (Burns et al., 1999). Therefore, we used aparallel-plate flow chamber and adapted continuous flow-loop (Cytodyne)to establish a perpetual fluid environment of circulating monocytesallowing for the observation and quantification of the threecharacteristic events: tethering, rolling and firm adhesion (Kinashi,2005; Ley et al., 2007; Springer, 1995). Twenty-four hours after singledose of 30 Gy, RECs were placed in the flow chamber and interacting U937monocytic cells were observed and quantified via phase-contrastmicroscopy. As shown in FIG. 2B (lower panel A-C referring to Control,IR, IR+KZ-41, respectively), upon digital video analysis, rolling,tethering and firm adhesion of monocytes across REC monolayer wassignificantly enhanced following radiation compared to unirradiated RECs(FIG. 2B, upper panel; 2±2 (A) vs. 87±18 (B) adhered cells; P<0.05). Incontrast, treatment with KZ-41 in irradiated RECs significantly reducedU937 adherence (25±12 (C) vs. 87±18 (B) adhered cells; *P<0.05).Interestingly, rolling activity of monocytes was unaffected withtreatment of KZ-41 (observational data not shown).

We demonstrated herein that irradiation-induced ICAM-1 levels could bereduced by KZ-41 treatment. For confirmation that the surface-inductionof selectins (specifically P-selectin) was unchanged by treatment ofKZ-41 after radiation, we performed confocal microscopy on the same RECsplaced in the flow chamber experiments (FIG. 3A). In unirradiated RECs,the level of P-selectin was almost undetectable in stark contrast tovery prominent signals shown in irradiated RECs. Both in vitro and invivo models of radiation-induced vascular injury have established ICAM-1upregulation as an important pathological indicator of inflammation(Gaber et al., 2003). We examined ICAM-1 expression on the sameflow-chamber slides. Within 24 h following irradiation, ICAM-1 levels inirradiated RECs (IR) were increased over unirradiated RECs (control)(P<0.05). This increased level (IR) was significantly reduced in KZ-41treated RECs (IR+KZ-41) (P<0.05).

To rule out the possibility that KZ-41 treatment lead to a disruption inrate and/or extent of protein trafficking to cellular surface, wecollected cellular lysates of RECs irradiated, with or without treatmentof KZ-41 and probed for total ICAM-1 protein by immunoblotting. Thetotal amount of protein that accumulated in the 24 hours followingexposure to radiation both on the cell surface and in the cytosolicfraction (P<0.05 vs. unirradiated RECs) was reduced with treatment ofKZ-41 (P<0.05) (FIG. 3B).

We further demonstrated herein that KZ-41 regulated the ICAM-1expression levels through a p38^(MAPK)-dependent mechanism. We firstperformed a time-course experiment to examine the p38^(MAPK)phosphorylation status post irradiation. As shown in FIG. 4A, relativeto total p38 levels, phospho-p38 reached a transient plateau over fourto eight hours post-IR. We then collected and analyzed treated cells 4hours following irradiation. As shown in FIG. 4B, irradiated RECs thatwere treated with KZ-41 prior to radiation exposure (IR+KZ-41) had about30% reduction in phosphorylated p38^(MAPK) (irradiation) (P<0.05).

We further demonstrated herein that KZ-41 could increase the stabilityof p53 although KZ-41 did not cause a significant reduction inphosphorylation of p53 when normalized to total p53 protein. As shown inFIGS. 5A, 5B and 5C, after exposure to radiation, RECs treated withKZ-41 did not show any significant reduction in phosphorylation of p53when normalized to total p53 protein (P>0.05). However, when total p53was normalized to housekeeping protein, GAPDH, we saw a significantreduction in total p53 protein amount (FIG. 5D, *P<0.005, **P<0.05).KZ-41 could enhance the stability of p53 protein through theMDM2-mediated ubiquination pathway. RECs were treated with a p53-MDM2inhibitor, NCS622875 (RITA, 10 μM) that effectively blocks theinteraction between MDM2-p53 at the transactivation domain-bindingcleft, consequently promoting p53 accumulation (Espinoza-Fonseca, 2005;Parks et al., 2005; Vassilev et al., 2004; Zhong and Carlson, 2005).When compared to untreated RECs, RITA treatment for four hourssignificantly enhanced p53 accumulation (*P<0.05). In contrast,concomitant treatment with KZ-41 or SB202190 did not significantly alterthis occurrence (FIG. 5E, P>0.05).

We further demonstrated that KZ-41 could prevent the irradiation-inducedp38-p53-dependent apoptotic signaling pathway. We used two methods ofanalysis to detect the presence of cleaved caspase-3 in RECs followingirradiation: sandwich ELISA (FIG. 6A) and In-Cell Western (ICW) (FIG.6B). Results in both assays showed a significant increase in cleavedcaspase-3 24 hours after radiation (*P<0.05 ELISA and ICW). With KZ-41treatment, this level was significantly reduced (**P<0.005, **P<0.05;ELISA, ICW, respectively).

We further demonstrated that KZ-41 could reduce the irradiation-inducedmigratory potential of RECs by mitigating p38-dependent phosphorylationof focal adhesion scaffold protein paxillin. We examined if irradiatedRECs showed enhanced VEGF secretion, promoting apro-survival/pro-proliferative phenotype in the surviving fraction ofcells. As a result shown in FIG. 7A, 24 hours post-irradiation, wedetected significant increases in VEGF in culture media (*P<0.001). Inaddition, we examined the phosphorylation of paxillin in irradiated RECsand found a substantial increase of tyrosine phosphorylation of paxillin(Y118) 24 hours following irradiation (FIG. 7B; P<0.05). Further, weevaluated the effect of KZ-41 and SB202190 on paxillin phosphorylationin irradiated RECs and found that both KZ-41 and SB202190 significantlyreduced phosphorylation of paxillin at Y118 (FIG. 7B; #P<0.05). Stillfurther, we examined the effect of KZ-41 on the pathological RECpro-migratory/proliferative response that was triggered byradiation-induced VEGF expression and subsequentpaxillin-phosphorylation and found that KZ-41 could significantly reducethe proliferative phenotype to almost normal levels in irradiated RECs(FIG. 7C, *P<0.05, **P<0.05).

We herein further demonstrated that KZ-41 could pathologicalneovascularization and avascular areas in an oxygen-induced retinopathymouse model. KZ-41 was delivered to mouse retinal cells through adrug-delivery system that we formulated so that the drug could beapplied topically as an eye-drop. To confirm that efficacy of thismethod for delivering effective treatments to the back of eye, weperformed a pilot ocular-PK study using C57BL/6J adult male mice (n=3mice/time-point; FIG. 9). As shown in FIG. 8, nanoemulsion had aviscosity of 17 mPa·s, average particle size of 60 nm which wouldincrease to 75 nm after being placed at room temperature for 60 days,and pH of 6.5. As shown in FIG. 9, ocular pharmacokinetic analysisconfirmed that KZ-41 penetrated through the cornea tissue within 5minutes and produced a mean peak vitreous humor concentration of130.1±4.6 mg/mL at 15 minutes. KZ-41 concentration in the vitreous humordropped exponentially with a half-life of 2.2±0.4 h. The volume ofdistribution was 1.8±0.4 L/kg, clearance was 0.57±0.07 L/h/kg (FIG. 9).Systemic circulation was achieved 30 minutes after ocularadministration, correlating precisely with the apparent distribution ofKZ-41 out of the eye.

The murine oxygen-induced retinopathy (OIR) model is the well-known andindustry-recognized in vivo model for studying the effect of genomic orpharmacologic manipulation of key signaling proteins on the naturalhistory of VEGF-induced proliferative retinopathies (e.g., RR,retinopathy of prematurity, and proliferative diabetic retinopathy)(Connor et al., 2009; Smith et al., 1994). We used this model toevaluate the effects of KZ-41 on preventing VEGF-induced pathologicalretinal neovascularization (RNV) driven by oxidative stress and ischemicinjury. Specifically, mouse pups were exposed to 75% oxygen atpost-natal day 7 (P7) for 5 days and then returned to normal oxygen atP12. Mice received daily ocular administration of either KZ-41 (100mg/kg) or vehicle (ophthalmic NE) from P12 to P17 using the abovedelivery method. Eyes were enucleated at P17 and retinal whole-mountsstained for endothelial cells. Avascular area and vascular tufts werequantified as a percentage of total retinal vasculature using confocalmicroscopy (Connor et al., 2009).

As shown in FIGS. 10A, 10B, 10C, and 10D, we examined the flat-mountedretinas at P17 for each experimental group (A-D; Normoxia-N17,OIR17-untreated, OIR17+Vehicle, and OIR17+KZ-41.) Both OIR17 and OIR17+Vmice showed larger avascular area (AV area) surrounding the optic discas compared to N17 control mice (FIG. 10A, 20.5±1.8, 18.6±3.1 vs.4.4±1.1 AV area, P<0.005) and extensive neovascularization area (NVarea) compared to normoxia controls (FIG. 10B, 24.7±2.3, 22.3±1.4 vs.0.76±0.28 percent NV area, P<0.005). There was no significant differencein total AV or NV area between OIR17 and OIR17+V mice (FIGS. 10A and10B; P>0.05). OIR17+KZ-41 mice showed extensive physiologicalrevascularization towards the optic disc with significant decreases inneovascular tuft formation as well as a two-fold reduction in avasculararea as compared to OIR+V mice (OIR+KZ-41 vs. OIR+V 8.6 vs. 18.6 AV %area, 16.5 vs. 22.3 NV % area; #P<0.001, P<0.01 respectively).

While OIR+KZ-41 mice still showed significant increases in NV areacompared to normoxia controls, AV area quantification was statisticallyindistinguishable from normoxia mice (P>0.05). We show that DNA damagingradiation triggers the accumulation of phosphorylated p38 and p53 topromote transcription and expression of inflammatory genes and proteins.Our quinic-acid derivative, KZ-41 has shown to inhibit p38-p53 signalingmechanisms to attenuate the expression of ICAM-1 and reduce apoptoticsignaling in IR-RECs.

Therefore, in this Example 1, we demonstrated that KZ-41 could helppreserve the integrity and functionality of the RECs, and prevent ordecrease the risk of monocyte adhesion, subsequent endothelial celldysfunction, neovascularization, and/or blindness. These effects wereseen in both in vitro and in vivo pre-clinical models radiationretinopathy.

Example 2 KZ-41 could Regulate Retinal Endothelial Cell Viability inConnection with Diabetic Retinopathy

In this Example 2, we demonstrated that KZ-41 could protect hRECs fromglucose-induced apoptosis. We showed that IGF-1R activity and thecascade PI3/Akt pathway were stimulated by KZ-41 in RECs. Therefore,KZ-41 could be a novel therapeutic agent for diabetic retinopathy.

The materials and methods used in this Example 2 are as follows.

Reagents.

KZ-41 was synthesized in Dr. Duane Miller's laboratory and verified tobe >96% pure by nuclear magnetic resonance spectroscopy (Zeng et al.,2009). Total IGF-R1, IRS-1, p-85, and Akt and phosphorylated(Tyr1135/1136) IGF-R1, (Ser473) Akt and (Tyr458) p85 primary, and GAPDHantibody (rabbit) primary antibodies were obtained from Cell Signaling(Danvers, Mass.). Secondary goat anti-rabbit IgG antibodies (IRDye800CW) were purchased from LI-COR Biotechnology (Lincoln, Nebr.). TheClass IA PI3 kinase inhibitor, LY294002, was kindly provided by Dr.Ramesh Ray (UT). D-Mannitol and Glucose were perched from Sigma.

Cell Culture.

Primary human retinal microvascular endothelial cells (REC, Lot 181)were acquired from Cell Systems Corporation (CSC, Kirkland, Wash.).Cells were grown in M131 medium containing microvascular growthsupplements (Invitrogen, Carlsbad, Calif.), 10 μg/mL gentamicin, and0.25 μg/mL amphotericin B. Prior to each experiment, cells weretransferred to high (25 mM) or normal (5 mM) glucose, or mannitol (25mM) medium for three days. Only primary cells within passage six wereused. Cells were quiesced by incubating in high or normal glucose mediumwithout fetal bovine serum for 24 hours and used to perform theexperiments unless otherwise indicated. To investigate cell-signalingthrough PI3 kinase, LY294002 was added to culture medium for 3 hr priorto treatment with or without KZ41 (10 μM) for 2 hr.

Caspase-3 Activity.

The Pathscan Cleaved Caspase-3 sandwich ELISA kit (Cell Signaling) wasused to evaluate endogenous cleaved caspase-3 levels in RECs accordingto the manufacturer's instructions. Cleaved caspase 3 level in normalglucose (5 mM), high osmolar (25 mM mannitol) or high glucose (25 mm)was detected following treatment with or without KZ-41 (10 μM) for 2 h.For all ELISA analyses, equal protein amounts were loaded into eachwell, allowing for comparisons using optical density (O. D.)

Western Blotting Analysis.

Cellular proteins were analyzed by Western blot after SDS-PAGE usingrabbit anti-human specific primary antibodies. Retinal endothelial cellswere ringed with cold phosphate-buffered saline after 2 hr KZ-41treatment. REC lysates were collected in modified RIPA lysis buffer (20mM Tris, 2.5 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 40 mMNaF, 10 mM Na4P2O7, and 1 mM PMSF) with protease/phosphatase inhibitor(1×) cocktail (Roche; Indianapolis, Ind.). Lysates were kept on ice for30 minutes followed by centrifugation to remove insoluble materials(12,000 g at 4° C. for 30 minutes). Total protein was measured using BCAassay (Pierce, Rockford, Ill.). Protein samples (10˜50 μg) mixed with4×LDS loading buffer with 2.5% 2-mercaptoethanol (Sigma), heated to 70°C. for 10 minutes, and loaded on NuPAGE 4-12% Bis-Tris gel (Invitrogen,Carlsbad, Calif.). Immunoblotting was performed with nitrocellulosemembranes (Bio-Rad) at 170-mA start and 110-mA end at 25 V for 2 h inNuPAGE transfer buffer (Invitrogen) containing 20% methanol. Membraneswere blocked using Odyssey blocking buffer (LICOR) for one hour at roomtemperature with gentle shaking Membranes were then incubated at 4° C.with specific primary antibodies (1:1000) overnight. Cellular proteinwas normalized using GAPDH [1:10,000] (Cell Signaling). The secondaryantibody (IRDye 800CW goat anti-rabbit) [1:10,000] was incubated in thedark at room temperature for 45 minutes. Dual-channel infrared scan andquantitation of immunoblots were conducted using the Odyssey® Sainfrared imaging system with Image Studio (Ver. 3.1.4) (LI-COR, Lincoln,Nebr.).

Statistical Analysis.

All data in the different experimental groups are expressed as mean±S.D.and obtained from at least three independent experiments. Analysis ofvariance (ANOVA) was used to assess the statistical significance of thedifferences between groups, followed by Duncan's multiple-range test orStudent's t-test, where appropriate. A P value of <0.05 was consideredsignificant.

We herein demonstrated that KZ-41 could reduce glucose-induced apoptosisin RECs. Apoptotic cell death is triggered in RECs continuously exposedto high glucose concentrations (Costa et al., 2012; el-Remessy et al.,2005; Ho et al., 2000; Zhang et al., 2013). Activated (cleaved)caspase-3, a crucial effector of the terminal or execution phase of theapoptotic pathway, has been recognized as a reliable phenotypic markerof apoptosis (Zhang et al., 2005). Here, we evaluated the effect ofKZ-41 (10 μM) on caspase-3 activation in RECs cultured in either normalglucose (5 mM), high glucose (25 mM), or mannitol (25 mM) for threedays. As shown in FIG. 11, caspase-3 levels in RECs exposed to highglucose were significantly higher when compared to RECs cultured innormal glucose (0.62±0.04 vs. 0.29±0.05; P<0.01). RECs cultured inmannitol (25 mM), as an osmotic control, showed no increase in cleavedcaspase-3 activity when compared to RECs cultured in normal glucose(0.32±0.03 vs. 0.29±0.05; P>0.05). KZ-41 significantly reduced cleavedcaspase-3 levels RECs exposed to high glucose (0.37±0.09 vs. 0.62±0.04;P<0.01). Cleaved caspase-3 levels were unaltered by KZ-41 in RECscultured in either normal glucose or mannitol. Together, these resultsindicated that KZ-41 reversed glucose-induced caspase-3 activationwithout affecting constitutive caspase-3 levels in RECs.

We further demonstrated that Akt activity could be increased by KZ-41.Prolonged high glucose exposure inactivates the PI3-kinase/AKTpro-survival signaling pathway leading to reduced REC cell viabilitywithin three days (Costa et al., 2012; Jiang et al., 2012; Zhang et al.,2013). In this series of experiments, we evaluated total andphosphorylated (Ser473) Akt levels, as a readout of pro-survivalsignaling, over four days in RECs exposed to high glucose (25 mM).Phosphorylated Akt levels modestly increased over the first 18 hoursafter glucose exposure, but were significantly attenuated at 24-96 hours(FIG. 12A). Total Akt levels were unchanged over the four day period ofhigh glucose exposure. Consequently, the ratio of phosphorylated Akt tototal Akt protein expression was significantly decreased between 24-96hours.

In addition, we measured the effect of KZ-41 on total and phosphorylated(Ser473) Akt expression in RECs cultured in high glucose for 72 hours(FIG. 12B). Following 2 hours incubation, KZ-41 (10 μM) reversed theeffect of high glucose on phosphorylated Akt expression (1.13±0.16 vs.0.79±0.06; P<0.05) without altering total Akt expression. The net effectof KZ-41 treatment was restoration of the ratio of phosphorylated tototal Akt expression found in RECs cultured in normal glucose. The ratioof phosphorylated to total Akt expression was not significantly affectedby KZ-41 in RECs cultured in either normal glucose or mannitol.

We further demonstrated that KZ-41 could enhance PI3K activity.Phosphatidylinositide 3-kinase (PI3K) promotes pro-survival signaltransduction by phosphorylating Akt (Alessi et al., 1996). We used thePI3K inhibitor LY294002 to examine the requirement for PI3K in KZ-41'spro-survival signal transduction mechanism. Pretreatment of RECs withLY294002 resulted in a complete block of KZ-41-induced Aktphosphorylation suggesting KZ-41 signaling required PI3K (FIG. 13A).LY294002 inhibits class IA, class IB, and class III PI3Ks with equalpotency (Vanhaesebroeck et al., 2001). PI3K class IA comprises an 85 kDaregulatory subunit (p85) and a 110 kDa catalytic subunit (p110)(Carpenter et al., 1990). High glucose suppresses p85 phosphorylationand PI3K activity in RECs (references). Thus, we next examined theeffect of KZ-41 on glucose-induced alterations in p85 phosphorylation(Tyr458) (FIG. 13B). Following two hours incubation, KZ-41 (10 μM)reversed the effect of high glucose on phosphorylated p85 expression(0.995±0.086 vs. 0.687±0.008; P<0.01) without altering total p85expression. The net effect of KZ-41 treatment was restoration of theratio of phosphorylated to total p85 expression found in RECs culturedin normal glucose. These data suggest that KZ-41 exerted itspro-survival effects through PI3K class IA proteins.

We further demonstrated that KZ-41 could enhance IRS-1 expression. Wemeasured IRS-1 expression in RECs cultured in high glucose for 72 hours(FIG. 14). High glucose dramatically decreased IRS-1 expression(0.14±0.1 vs. 1.00±0.21; P<0.01). However, KZ-41 was able to partiallyreverse the impact of high glucose on IRS-1 expression (0.58±0.15 vs.0.14±0.1; P<0.05). These data suggest that the effect of KZ-41 ismediated, in part, through IRS-1.

We further demonstrated that KZ-41 treatment could activateIGF-1R-mediated survival signaling pathways in response to HG in RECcells. KZ-41 could enhance IGF-R1 activation and lead to increasedIRS-1/PI3K/Akt signaling. We measured the expression of phosphorylatedIGF-R1, total IGF-R1, and GAPDH proteins in REC cells cultured in normalglucose, mannitol, and high glucose medium with or without KZ-41treatment (FIG. 15A). High glucose inhibited phosphorylated(Tyr1135/1136) IGF-R1 (FIG. 15D) and total IGF-R1 expression (FIG. 15C)as well as the ratio of phosphorylated (Tyr1135/1136) to total IGF-R1(FIG. 15B). Whereas, the ratio of phosphorylated (Tyr1135/1136) to totalIGF-R1 in KZ-41-treated (10 μM, two hours) RECs was higher than thatfound in RECs cultured in normal glucose (1.31±0.10 vs. 1.00±0.05;P<0.05) or high glucose alone (1.31±0.10 vs. 0.70±0.05; P<0.01) (FIG.15B). Altogether, these data showed that KZ-41 could induceIGF-1R-mediated pro-survival signaling in RECs cultured in conditionsdesigned to mimic the diabetic milieu.

In summary, in the Example 2, we showed that KZ-41 could be a protectiveagent against high-glucose induced apoptosis in REC cells. We observedthat high glucose induced REC cells apoptosis. Apoptosis is defined asthe process of cell death associated with caspase activation orcaspase-mediated cell death. It is a necessary component of developmentand characteristic of all self-renewing tissues. Here, we demonstratedthat treatment with KZ-41 inhibited high glucose induced apoptosis ofREC cells, indicating an anti-apoptotic role for KZ-41 in REC cells.Therefore, KZ-41 could be used as a novel therapeutic candidate fordiabetic retinopathy.

Example 3 KZ-41 could Regulate Retinal Endothelial Cell Viability inConnection with Chemotherapy for Retinoblastoma

We previously found that exposure to a retinoblastoma cell cidalmelphalan dose (4 μg/mL) (Steinle et al., 2012) produced a greater than6-fold increase in REC death. In this Example 3, we further demonstratedthat KZ-41 could inhibit REC apoptosis by regulating the NF-κB pathway.

The materials and methods used in this Example 3 are as follows.

Reagents.

Melphalan was bought from Bioniche Pharma (Lake Forest, Ill.). KZ-41 wassupplied by Dr. C. Ryan Yates. ICAM-1 ELISA kit was purchased fromMillipore (Bilerica, Mass.). Cell death ELISA kit was purchased fromRoche Applied Science (Indianapolis, Ind.). NF-κB, phospho-NF-κB (S536),p38 MAPK, phospho-p38 MAPK (T180/Y182) antibodies and SB202190 (p38 MAPKinhibitor, blocks P38α and P38β) were purchased from Cell Signaling(Lake Placid, N.Y.). Actin antibodies were purchased from Santa CruzBiotechnology (Santa Cruz, Calif.). Lipofectamine™ RNAiMAX TransfectionReagent was purchased from Invitrogen (Carlsbad, Calif.). Human Sc siRNA(ON-TARGET plus nontargeting Pool D-001810-10), human ICAM-1 siRNA(ON-TARGET plus SMARTpool L-003502-00-0005), human TNF-α siRNA(ON-TARGET plus SMARTpool L-010546-00-0005), and human NF-κB siRNA(ON-TARGET plus SMARTpool L-003520-00-0005) were purchased fromDharmacon RNAi Technologies (Chicago, Ill.). Etanercept was obtainedfrom Dr. Arnold Postlewaite. Secondary anti-mouse and anti-rabbitantibodies conjugated with horseradish peroxidase were purchased fromPromega (Madison, Wis.). ECL for immunoblot development and signaldetection was purchased from Amersham Biosciences (Piscataway, N.J.,USA).

Cell Culture.

REC were provided by Cell System Corporation (CSC, Kirkland, Wash.) andgrown in Medium 131 containing microvascular growth supplements (MVGS),10 μg/mL gentamycin, and 0.25 μg/mL amphotericin B. Cultures weremaintained at 37° C. in a humidified 95% air and 5% CO₂ atmosphere. Onlyprimary cells within passages 6 were used. RECs were growth-arrested byincubating in Medium 131 for 24 hours and used to perform theexperiments unless otherwise indicated. Y79 retinoblastoma cells werepurchased from ATCC. Cells were grown in suspension in RPMI medium withantibiotics and 20% fetal bovine serum. Cells were starved overnightbefore any treatments.

Cell Death Assay.

Equal number of RECs were placed into the 96-well plates and cultured to90% confluence. Cells were starved without growth factor overnight andtreated with the drug for 24 hours. Cells were washed with PBS twice andresuspended in 200 μL lysis buffer, incubated for 30 minutes at roomtemperature. Lysates were centrifuged at 200×g for 10 minutes, and 20 μLof cell lysates were transferred into the streptavidin-coated MP undergentle shaking for 2 hours at 20° C. Supernatants were removed and thewells were washed with incubation buffer. ABTS solution was added todevelop color detected at 405 nm (vs. 490 nm reference).

ICAM-1 ELISA.

An ELISA for ICAM-1 level was performed using an ICAM-1 ELISA assay kitaccording to the manufacturer's instructions to evaluate the ICAM-1level following treatment with melphalan and KZ-41, ICAM-1 siRNA, TNF-αsiRNA, NF-κB siRNA, or Enbrel (10 uM). For all ELISA analyses, equalprotein amounts were loaded into each well, allowing for comparisonsusing optical density (O. D.).

Western Blotting.

After appropriate treatments and rinsing with cold phosphate-bufferedsaline, REC were lysed in the lysis buffer containing the protease andphosphatase inhibitors and scraped into the tubes. Equal amounts ofprotein from the cell or tissue extracts were separated on the pre-casttris-glycine gel (Invitrogen, Carlsbad, Calif.), blotted onto anitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer,pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, the membrane wastreated with NF-κB, Phospho-NF-κB, p38^(MAPK), Phospho-p38^(MAPK)antibodies (1:500) followed by incubation with horseradish peroxidaselabeled secondary antibodies. The antigen-antibody complexes weredetected using chemilluminescence reagent kit (Thermo Scientific).

Transfections.

REC were transfected with ICAM-1 siRNA, TNF-α siRNA, or NF-κB siRNA at afinal concentration of 20 nM using Lipofectamine™ RNAiMAX TransfectionReagent according to the manufacturer's instructions. Aftertransfection, cells were starved in MVGS-free Medium 131 for 24 hoursand used as required.

Statistics.

All the experiments were repeated in triplicate, and the data arepresented as mean±SEM. Data was analyzed by Kruskal-Wallisnon-parametric test followed by Dunn's test with p-values <0.05considered statistically significant. In the case of Western blotting,one representative blot is shown.

We demonstrated that KZ-41 could inhibit melphalan-induced RECapoptosis. We have previously found that 4 μg/ml melphalan increases RECcell death (Steinle et al., 2012). We herein verified that 4 μg/mL didincrease DNA fragmentation (FIG. 16A). To our surprise, KZ-41 treatmentcould counter the pro-apoptotic effects of melphalan. As shown in FIG.16A, the effect of KZ-41 on inhibiting melphalan-induced REC apoptosiswas dose-dependent. A significant decrease of apoptosis in comparison tothat in RECs treated with melphalan alone was seen when KZ-41 was usedto treat RECs at a concentration of 10 μM (see the bar over the group“KZ 10 μM+MeI” and the bar over the group “Melphalan” in FIG. 16A).

We further demonstrated that KZ-41 did not affect apoptosis of Y79retinoblastoma cells. While KZ-41 could inhibit melphalan-induced RECapoptosis, KZ-41 did not inhibit the death of retinoblastoma cells. Wetested various doses of melphalan on Y79 retinoblastoma cells and found4 μg/mL produced maximal apoptosis (FIG. 16B). KZ-41 did not affectapoptosis of the Y79 cells because there was no significant differenceof apoptosis of Y79 cells between the group treated with melphalan aloneand the group treated with both melphalan and KZ-41 (FIG. 16C) (P>0.05).

We further demonstrated that KZ-41 could inhibit melphalan-inducedICAM-1 expression levels in RECs. We have previously reported thatmelphalan increases ICAM-1 mRNA and protein levels (Steinle et al.,2012). We performed an ICAM-1 ELISA after KZ-41+melphalan treatment inRECs. As shown in FIGS. 17A and 17B, when 10 μM KZ-41 was added 30minutes before melphalan treatment, we saw significantly decreasedICAM-1 levels in RECs. Therefore, KZ-41 could block melphalan-inducedICAM-1 levels in RECs.

We further demonstrated that TNF-α did not decrease ICAM-1 levels inRECs. We examined whether inhibition of TNF-α with TNF-α siRNA oretanercept, a TNFα receptor antagonist, could reduce melphalan-inducedincreased ICAM-1 levels in RECs. As result, neither TNF-α siRNA noretanercept could reduce ICAM-1 levels (FIGS. 18A and 18B). Whileetanercept did reduce REC apoptosis after melphalan treatment, it didnot reach statistical significance (FIG. 18C).

We further demonstrated that KZ-41 could inhibit melphalan-inducedICAM-1 activation through NF-κB. We found that Melphalan (4 μg/mL) couldinduce NF-κB phosphorylation in a time-dependent manner in RECs (FIG.19A). Maximum increases in NF-κB^(Ser536) phosphorylation occurred at 30minutes, with increased NF-κB levels for at least 2 hours (FIG. 19A). Inaddition, melphalan-induced ICAM-1 upregulation was blocked by eitherKZ-41 (FIG. 19B, comparing the bar in the group treated with bothmelphalan and KZ-41 with the bar in the group treated with melphalanalone) or NF-κB siRNA (FIG. 19D, comparing the bar in the group treatedwith both melphalan and NF-κB siRNA with the bar in the group treatedwith melphalan and Sc siRNA). As shown in FIG. 19C, demonstrates theNF-κB siRNA significantly reduce NF-κB levels.

We further demonstrated that KZ-41 could inhibit melphalan-inducedICAM-1 activation through p38^(MAPK) in RECs. We first found that KZ-41could restore the phosphorylation level of P38^(MAPK) that was inducedby melphalan in RECs. See FIG. 20A, comparing the bar over the group ofMel+KZ-41 with the bar over the group of MeI. We then tested whetherKZ-41 could still inhibit melphalan-induced up-regulation of ICAM-1levels if p38^(MAPK) was blocked by SB202190 (a p38^(MAPK) inhibitor),and found that KZ-41 could not inhibit melphalan-induced up-regulationof ICAM-1 levels if p38^(MAPK) was blocked by SB202190 (FIG. 20B),suggesting that KZ-41's inhibitory effects on melphalan-induced ICAM-1activation depended on p38^(MAPK) in RECs.

We further demonstrated that KZ-41 could inhibit melphalan-induced RECapoptosis through p38^(MAPK). We used NF-κB siRNA, the p38^(MAPK)inhibitor SB202190, and ICAM-1 siRNA to in REC apoptosis analysis. Wedemonstrated that ICAM-1 siRNA had inhibitory effects on ICAM-1expression (FIG. 21A), that ICAM-1 siRNA could counter melphalan'sproapoptotic effects in RECs (FIG. 21B), and that KZ-41 could notinhibit melphalan-induced REC apoptosis if p38^(MAPK) was blocked bySB202190, suggesting that KZ-41's inhibitory effects onmelphalan-induced apoptosis depended on p38 in RECs (FIG. 21C).

In summary, we demonstrated that KZ-41 could protect the REC frommelphalan-induced REC toxicity. Super selective intra-ophthalmic arterychemotherapy (SSIOAC) using melphalan continues to be used for thetreatment of retinoblastoma (Abramson et al., 2008; Gobin et al., 2011),despite the reports of deleterious changes to the retina and choroid ofchildren (Ditta et al., 2012; Shields et al., 2011; Wilson et al.,2011). One option to improve this therapy is to mitigate the directmelphalan-induced REC toxicity. Here we demonstrated that KZ-41 couldprotect the REC, while not preventing melphalan-induced apoptosis in Y79retinoblastoma cells. Indeed, KZ-41 was effective in preventingmelphalan-induced apoptosis in REC, without altering melphalan's actionson retinoblastoma cells and could serve as an agent for preventing,mitigating, or cure the rental toxicity seen in SSIOAC.

While the present invention has been disclosed with reference to certainembodiments, numerous modifications, alterations, and changes to thedescribed embodiments are possible without departing from the full scopeof the invention, as described in the specification and claims.

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We claim:
 1. A method for regulating retinal endothelial cell viabilityin treating radiation retinopathy in a mammal, comprising administeringto the mammal a therapeutically effective amount of a quinic acid analoghaving a structure as in

where the ring may be singly, doubly, or completely saturated; R¹ and R²are each independently H, straight or branched alkyl, aryl, benzyl,arylalkyl, or heterocyclic amine; R³ may be present or absent and, ifpresent, may be H, hydroxyl, ether, alkoxy, or aryloxy; and R⁴, R⁵, andR⁶ are each independently H, hydroxyl, and alkoxy.
 2. The method ofclaim 1 wherein R¹ and R² of Formula I form a piperidine ring withNitrogen.
 3. The method of claim 1 wherein when one of R¹ or R² ofFormula I is hydrogen, the other of R¹ or R² is alkyl.
 4. The method ofclaim 3 wherein the alkyl is —C3H7 and each of R³-R⁶ is hydroxyl.
 5. Themethod of claim 1, wherein one or more of R³, R⁴, R⁵ or R⁶ are eachindependently connected to an antioxidant through an ester bond.
 6. Themethod of claim 5, wherein the antioxidant is selected from the groupconsisting of caffeic acid, ferulic acid, and sinapic acid.
 7. Themethod of claim 1, wherein the radiation retinopathy is due to exposureof the mammal's retinal endothelial cells to radiation.
 8. The method ofclaim 7, wherein the radiation is used to treat an intraocular tumor. 9.The method of claim 7, wherein the radiation is gray.
 10. The method ofclaim 1, wherein the mammal is a human.
 11. The method of claim 1,wherein the regulation of retinal endothelial cell viability is topromote or maintain retinal endothelial cell viability.
 12. The methodof claim 1, wherein the regulation of the retinal endothelial cellviability is to reduce retinal endothelial cell death.
 13. The method ofclaim 1, wherein the regulation of retinal endothelial cell viability isto prevent or reduce retinal neovascularization.
 14. The method of claim1, wherein the method is used in combination with one or more existingtreatment method for radiation retinopathy, diabetic retinopathy, ormitigating side effects on retinal endothelial cells in treatingretinoblastoma.
 15. The method of claim 1, wherein the quinic acidanalog is formulated in nanoemulsion.
 16. The method of claim 15,wherein the quinic acid analog is delivered as an eye-drop.